Hsv-1 oncolytic virus therapies that specifically kill alt dependent cancers

ABSTRACT

Recombinant herpes simplex virus (HSV)-1 capable of selectively replicating in alternative lengthening of telomeres (ALT)-dependent tumor cells are described. The recombinant HSV-1 are ICP0-deficient, such as by complete deletion of the ICP0 gene, or mutation of the ICP0 gene sufficient to diminish or eliminate E3 ubiquitin ligase activity of ICP0. In some cases, the recombinant HSV-1 further include additional gene deletions or mutations, such as those that render the virus glycoprotein C (gC) deficient, or include a heterologous gene, such as a gene encoding an immunostimulatory molecule. Methods of treating ALT-dependent cancer, and methods of selectively killing ALT-dependent tumor cells are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/169,421, filed Oct. 24, 2018, which is a continuation ofInternational Application No. PCT/US2017/029683, filed Apr. 26, 2017,published in English under PCT Article 21(2), which claims the benefitof U.S. Provisional Application No. 62/327,596, filed Apr. 26, 2016. Theabove-listed applications are herein incorporated by reference in theirentirety.

FIELD

This disclosure concerns recombinant herpes simplex viruses thatspecifically replicate in alternative lengthening of telomeres(ALT)-dependent cancer cells and their use for the treatment ofALT-dependent cancer.

BACKGROUND

Telomeres are specialized DNA repeats at the ends of chromosomes thatare replicated by the telomerase enzyme complex. In almost all normalcells with the exception of stem cells, telomerase activity is shut off.As a consequence, telomeres become progressively shorter with everyround of cellular DNA replication. When telomeres become criticallyshort, they trigger a cellular DNA damage response that inducesirreversible replicative arrest (Shay et al., Science 336, 1388-1390,2012). Thus, telomere shortening is a critical tumor suppressormechanism that limits the number of times a cell can replicate anddivide.

Cancer cells have limitless replicative potential due to their abilityto elongate and maintain their telomeres through one of two mutuallyexclusive mechanisms. In the majority of human cancers, human telomerasereverse transcriptase (hTERT) is upregulated. However, in approximately10% of human cancers, telomere length is maintained through a homologousrecombination-based mechanism known as the alternative lengthening oftelomeres (ALT) (Cesare and Reddel, Nat Rev Genet 11, 319-330, 2010).The strict dependence of replication on telomere maintenance makes it anattractive target for cancer therapy. However, treatment of mouse tumormodels with telomerase inhibitors leads to a switch and outgrowth ofALT-dependent tumors (Hu et al., Cell 148, 651-663, 2012). Therefore, iftargeting of telomere maintenance is going to be a viable cancertherapeutic option, it is imperative to be able to target telomerase aswell as ALT-dependent cancers. Despite nearly two decades since theidentification of ALT, there are currently no rational targets ortherapies for ALT-dependent tumors.

SUMMARY

Provided herein are methods of treating an alternative lengthening oftelomeres (ALT)-dependent cancer in a subject. The methods can includeselecting a subject having an ALT-dependent cancer; and administering tothe subject a recombinant herpes simplex virus (HSV)-1 that is infectedcell protein 0 (ICP0)-deficient, glycoprotein C (gC)-deficient, or bothICP0-deficient and gC-deficient, thereby treating the ALT-dependentcancer in the subject. In some embodiments, the ICP0-deficient HSV-1 hasa disruption in the ICP0 gene that diminishes or eliminates expressionof functional ICP0. In some examples, the disruption in the ICP0 geneincludes a complete deletion of the ICP0 gene, a partial deletion of theICP0 gene, an insertion in the ICP0 gene and/or a point mutation in theICP0 gene. In some embodiments, the HSV-1 has a modification of the gCgene that diminishes or eliminates expression of gC, or that results inexpression of a truncated gC. In some cases, the recombinant HSV-1includes disruptions in one or more additional viral genes or includes aheterologous gene, such as a gene encoding an immunostimulatorymolecule.

Also provided herein is a recombinant HSV-1 having a complete deletionof the ICP0 gene, and a heterologous gene encodinggranulocyte-macrophage colony-stimulating factor (GM-CSF). Furtherprovided is a recombinant HSV-1 having a complete deletion of the ICP0gene, a partial deletion of the ICP47 gene, and a heterologous geneencoding GM-CSF. Use of the recombinant viruses for the treatment ofALT-dependent cancer is also provided by the present disclosure.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G: (FIG. 1A) Model for the restriction of herpes simplex virus(HSV)-1 replication by promyelocytic leukemia (PML) nuclear bodies(NBs). (FIG. 1B) BJ cells were infected with the indicated virus andincubated for 24 hours post-infection (hpi). Cells were fixed andstained for HSV infected cell polypeptide 4 (ICP4) and PML and the edgeof newly forming plaques was imaged on a Zeis LSM 780 confocalmicroscope at 63× magnification. The viral protein ICP4 marks incomingviral genomes and shows co-localization between PML NBs and viralgenomes in the absence of infection cell polypeptide 0 (ICP0). Scale baris 10 μm. (FIG. 1C) Model for the role of PML NBs in ALT cells. (FIG.1D) Immunofluorescence-fluorescence in situ hybridization (IF-FISH) ofU2OS cells for telomeric C-rich repeats, which are labeled with apeptide nucleic acid (PNA)-TelC FISH probe and anti-PML antibody. Scalebar is 10 μm. (FIG. 1E) 1E5 BJ cells were infected with either HSV-1wild-type (WT) or HSV-1 ΔICP0 at the indicated multiplicity of infection(MOI) and incubated for 36-48 hours. Plates were stained with crystalviolet and plaques were counted. Wells that were completely lysed wereconsidered to have had 100% infection. (FIG. 1F) Plaque assay of U2OScells infected with HSV-1 WT or HSV-1 ΔICP0 and analyzed as above. (FIG.1G) U2OS cells were infected with HSV-1 ΔICP0 or left uninfected. At36-48 hpi, cells were fixed and hybridized with the PNA TelC FISH probefollowed by IF staining for PML and ICP4. Cells were imaged at 63× onZeiss LSM 780 confocal microscope. Scale bar is 10 μm.

FIGS. 2A-2J: (FIG. 2A) Primary small airway epithelial cells (SAEC) wereinfected with HSV-1 ΔICP0 or HSV-1 WT virus at an MOI=1. At 8 hpi, RNAwas collected and subjected to RNA sequencing. Differentially expressedgenes between HSV-1 ΔICP0 and HSV-1 WT infected cells are plotted on avolcano plot with those genes that are significantly upregulated in theabsence of ICP0 shown to the right of the double vertical lines andthose significantly downregulated in the absence of ICP0 shown to theleft of the double vertical lines. Significance was determined by ap-value ≤0.05 and Log2 fold change ≥0.8 or ≤−0.8. (FIG. 2B) Ahypergeomteric test was performed using MSigDB to calculate the overlapbetween the hallmark gene set from MSigDB and those genes significantlyupregulated in the absence of ICP0. The top ten overlapping pathways areshown with corresponding p-value. (FIG. 2C) Overlap between the curatedgene sets in MSigDB and those genes upregulated in the absence of ICP0.The top ten overlapping pathways are shown with corresponding p-value.(FIG. 2D) The ALT cell line U2OS were infected with HSV-1 ΔICP0 or HSV-1WT at an MOI=1. At 12 hpi, RNA was collected and subjected to microarrayanalysis on an affymetrix human Genechip 1.0 ST array. Differentiallyexpressed genes were plotted on a volcano plot with the significantlyupregulated genes shown on the upper right and significantlydownregulated genes shown on the upper left. Significance was determinedby a p-value ≤0.05 and Log2 fold change ≥0.8 or ≤−0.8. (FIG. 2E) Venndiagram showing overlap between the genes significantly suppressed byICP0 in SAEC and U2OS cells. (FIG. 2F and FIG. 2G) SAOS2 (ALT) and SKBR3(ALT) cells were infected and RNA from these cells was analyzed bymicroarray as described for U2OS cells. (FIG. 2H and FIG. 2J) Thetelomerase positive cell lines HOS and SJSA1 were infected at MOI=1 withHSV-1 ΔICP0 or WT. RNA was collected 12 hpi and analyzed by microarray.Differentially expressed genes are plotted as a volcano plot.Significance was determined by a p-value ≤0.05 and Log2 fold change≥0.58 or ≤−0.58 for HOS and Log2 fold change ≥1 or ≤−1 for SJSA1 cells.(FIG. 2I) Overlap between the hallmark gene set from MSigDB and thosegenes upregulated in the absence of ICP0. The top ten overlappingpathways are shown with corresponding p-value.

FIGS. 3A-3D: (FIG. 3A) Indicated cells were treated with varying amountsof recombinant interferon (IFN)-α for 4 hours at which point RNA washarvested from the cells. Fold change relative to the untreated controlfor indicated genes was determined by reverse transcriptase-quantitativepolymerase chain reaction (RT-qPCR) using the ΔΔCq method and GAPDH fornormalization. Error bars represent the standard error of the mean(SEM). (FIG. 3B) Cells were transfected with an empty plasmid, shearedcalf thymus DNA or transfection reagent only and incubated for 6 hours.RNA was collected and fold change relative to untransfected cells wasdetermined by RT-qPCR. (FIG. 3C) HOS or U2OS cells were infected withHSV-1 WT or HSV-1 ΔICP0 at an MOI=1 for 4 hours following which RNA wascollected and analyzed by RT-QPCR. (FIG. 4D) HOS or U2OS cells weretransfected with 2 μg of PolyI:C or empty plasmid. At 4 hourspost-transfection, RNA was harvested and subjected to RT-qPCR using 18sfor normalization.

FIGS. 4A-4B: (FIG. 4A) Indicated cells were plated in 24 well plates andinfected in duplicate with either HSV-1 WT or HSV-1 ΔICP0 at anexperimentally determined MOI that would yield ˜30 plaques/well in theHSV-1 WT infected wells. At 36-48 hours post-infection, cells werestained with crystal violet and plaques were counted. The average numberof plaques in ΔICP0 infected cells divided by the average number ofplaques in WT infected cells was calculated to yield the replicationefficiency of HSV-1 ΔICP0 relative to HSV-1 WT in the indicated celltype, which was then normalized to 100%. Error bars represent standarddeviation (SD). (FIG. 4B) A model representing the required cellularchanges for a cell to transition to ALT. These changes phenocopy HSV-1ICP0 activity, which subsequently rescues replication of an ICP0-deletedHSV-1.

FIGS. 5A-5B: (FIG. 5A) BJ cells were transfected with the indicatedsiRNA. At 48 hours post-transfection, cells were harvested for RNA tomonitor knock down efficiency of PML, ATRX or DAXX. Fold-change relativeto siControl transfected BJ cells was determined by RT-qPCR using theΔΔCq method and GAPDH as a normalizer. (FIG. 5B) BJ cells weretransfected with indicated siRNA. At 48 hours post-transfection, cellswere infected with HSV-1 ΔICP0 or HSV-1 WT and a plaque assay wasperformed.

FIGS. 6A-6B: (FIG. 6A) Full panel of cells screened for sensitivity toIFN-α treatment as described in FIG. 3. Shown from left to right foreach dose of IFNα is the transcriptional induction of IFIT1, IFIT2,IFIT3, MX1, OAS1 and OAS2 and MX1. (FIG. 6B) Full panel of cellstransfected with empty plasmid DNA of calf thymus DNA as described inFIG. 3. Shown from left to right for each type of DNA is induction ofIFIT1, IFIT2, IFIT3, MX1, OAS1 and OAS2 and MX1 mRNA.

FIGS. 7A-7E: Oncolytic HSV-1 administration to nude mice harboringxenograft tumors stably transduced with a lentiviral vector expressing aCMV promoter-driven luciferase reporter gene. (FIG. 7A) Tumor cellviability, as measured by luciferase activity, following administrationof HSV-1 WT and HSV-1 ΔICP0 to mice harboring SAOS2 (ALT-dependent)tumor cells. Virus was administered at Day 0 (5×10⁵ PFU), Day 3 (5×10⁵PFU) and Day 8 (1×10⁶ PFU). (FIG. 7B) Tumor volume followingadministration of HSV-1 WT and HSV-1 ΔICP0 to mice harboring SAOS2(ALT-dependent) tumor cells. Virus was administered at Day 0 (5×10⁵PFU), Day 3 (5×10⁵ PFU) and Day 8 (1×10⁶ PFU). (FIG. 7C) Tumor cellviability following administration of HSV-1 WT and HSV-1 ΔICP0 to miceharboring SAOS2 (ALT-dependent) tumor cells. Virus was administered atDay 0 (5×10⁵ PFU), Day 3 (5×10⁵ PFU) and Day 5 (1×10⁶ PFU). (FIG. 7D)Tumor volume following administration of HSV-1 WT and HSV-1 ΔICP0 tomice harboring SAOS2 (ALT-dependent) tumor cells. Virus was administeredat Day 0 (5×10⁵ PFU), Day 4 (5×10⁵ PFU) and Day 8 (1×10⁶ PFU). (FIG. 7E)Tumor cell viability following administration of HSV-1 WT and HSV-1ΔICP0 to mice harboring A549 (Tel+) tumor cells. Virus was administeredat Day 0 (5×10⁵ PFU), Day 3 (5×10⁵ PFU) and Day 7 (1×10⁶ PFU).

FIG. 8: Schematic diagrams of HSV-1 WT, HSV-1 ΔICP0 GM-CSF and HSV-1ΔICP0/ΔICP47 GM-CSF.

FIG. 9A-9B: Indicated cells were plated in 24 well plates and infectedin duplicate with either HSV-1 WT or HSV-1 ΔICP0. At 36-48 hourspost-infection, cells were stained with crystal violet and plaques werecounted. The average number of plaques in ΔICP0 infected cells dividedby the average number of plaques in WT infected cells was calculated toyield the relative plaque forming efficiency of HSV-1 ΔICP0 relative toHSV-1 WT in the indicated cell type, which was then normalized to 100%.Error bars represent standard deviation (SD). FIG. 9A shows ALT cellline SJRH30 in comparison with U2OS. FIG. 9B shows ALT cell line G292 incomparison with U2OS.

FIG. 10 shows the sequence analysis of the HSV-1 gC gene from parentalHSV-1 WT (strain 17) and HSV-1 ΔICP0 (strain d11403). Nucleotidealignment of a fragment of the gC gene between HSV-1 WT (SEQ ID NO: 17)and HSV-1 ΔICP0 (SEQ ID NO: 18) is shown. Deletion is indicated by “−”.Numbering of nucleotides is indicated at the top. Also shown is thealignment of gC proteins based on DNA sequence results. The underlinedHSV-1 ΔICP0 gC sequence has no homology to the wild type gC protein (WT:SEQ ID NO: 19 from amino acid 60-70, SEQ ID NO: 20 from amino acid171-180; d11403: SEQ ID NO: 21 from amino acid 60-70, SEQ ID NO: 22 fromamino acid 171-174). Stop codon is indicated by “*”. Numbering of aminoacids is indicated above the sequences.

Sequence Listing

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, created on Oct. 30, 2020, 786 KB, which is incorporated byreference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the nucleotide sequence of the IFIT1 forward primer.

SEQ ID NO: 2 is the nucleotide sequence of the IFIT1 reverse primer.

SEQ ID NO: 3 is the nucleotide sequence of the IFIT2 forward primer.

SEQ ID NO: 4 is the nucleotide sequence of the IFIT2 reverse primer.

SEQ ID NO: 5 is the nucleotide sequence of the IFIT3 forward primer.

SEQ ID NO: 6 is the nucleotide sequence of the IFIT3 reverse primer.

SEQ ID NO: 7 is the nucleotide sequence of the OAS1 forward primer.

SEQ ID NO: 8 is the nucleotide sequence of the OAS1 reverse primer.

SEQ ID NO: 9 is the nucleotide sequence of the OAS2 forward primer.

SEQ ID NO: 10 is the nucleotide sequence of the OAS2 reverse primer.

SEQ ID NO: 11 is the nucleotide sequence of the MX1 forward primer.

SEQ ID NO: 12 is the nucleotide sequence of the MX1 reverse primer.

SEQ ID NO: 13 is the nucleotide sequence of the GAPDH forward primer.

SEQ ID NO: 14 is the nucleotide sequence of the GAPDH reverse primer.

SEQ ID NO: 15 is the nucleotide sequence of HSV-1 ΔICP0 GM-CSF.

SEQ ID NO: 16 is the nucleotide sequence of HSV-1 ΔICP0/ΔICP47 GM-CSF.

SEQ ID NO: 17 is the HSV-1 WT glycoprotein C (gC) nucleotide 181-200sequence.

SEQ ID NO: 18 is the HSV-1 strain d11403 gC nucleotide 181-199 sequence.

SEQ ID NO: 19 is the HSV-1 WT gC amino acid 60-70 sequence.

SEQ ID NO: 20 is the HSV-1 strain d11403 gC amino acid 60-70 sequence.

SEQ ID NO: 21 is the HSV-1 WT gC amino acid 171-180 sequence.

SEQ ID NO: 22 is the HSV-1 strain d11403 amino acid 171-174 sequence.

SEQ ID NO: 23 is the nucleotide sequence of HSV-1 ΔICP0/AgC GM-CSF.

SEQ ID NO: 24 is the nucleotide sequence of HSV-1 ΔICP0/ΔICP47/ΔgCGM-CSF.

SEQ ID NO: 25 is the amino acid sequence of HSV-1 ICP0 corresponding toGenBank Accession No. AEQ77030.1.

SEQ ID NO: 26 is the amino acid sequence of HSV-1 glycoprotein Ccorresponding to Uniprot P10228.

SEQ ID NO: 27 is the nucleotide sequence of wild-type HSV-1 glycoproteinC corresponding to EMBL-EBI identifier CAA32294.1.

DETAILED DESCRIPTION I. Abbreviations

ALT alternative lengthening of telomeres

APB ALT-associated PML NB

ECTR extrachromosomal telomeric repeat

hpi hours post-infection

HSV herpes simplex virus

FISH fluorescence in situ hybridization

GBM glioblastoma multiforme

ICP0 Infected cell protein 0

ICP4 infected cell protein 4

IF immunofluorescence

IFN interferon

ISG interferon stimulated gene

MOI multiplicity of infection

NB nuclear body

PML promyelocytic leukemia

PNA peptide nucleic acid

RT-qPCR reverse transcriptase-quantitative polymerase chain reaction

SAEC small airway epithelial cells

SD standard deviation

SEM standard error of the mean

TERT telomerase reverse transcriptase

TMM telomere maintenance mechanism

VECTR viral and extrachromosomal DNA transcriptional response

WT wild-type

II. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as atherapeutic agent (e.g. an oncolytic virus), by any effective route.Exemplary routes of administration include, but are not limited to,injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, intratumoral, and intravenous), oral, intraductal,sublingual, rectal, transdermal, intranasal, vaginal and inhalationroutes.

Alternative lengthening of telomeres (ALT): A mechanism used bymammalian cells for maintaining telomeres. Approximately 10% of tumorsuse ALT as a telomere maintenance mechanism (TMM), while the majority ofcancers are dependent on telomerase to maintain telomere length.

ALT-dependent cancer: Any type of cancer that relies on ALT formaintaining telomeres. In some embodiments, the ALT-dependent cancer ischaracterized as being telomerase-negative, having extrachromosomaltelomeric repeat (ECTR) DNA, having altered PML bodies, permissive forinfection by ICP0-deficient HSV-1 and/or having a VECTR-deficientresponse. In some embodiments herein, the ALT-dependent cancer is a softtissue sarcoma, such as but not limited to, a pleomorphic sarcoma, afibrosarcoma, a leiomyosarcoma, a liposarcoma, an angiosarcoma, anepithelioid sarcoma or a chondrosarcoma. In other embodiments, theALT-dependent cancer is a cancer of the central nervous system, such asbut not limited to an astrocytoma (for example, a diffuse astrocytoma oran anaplastic astrocytoma), glioblastoma multiforme (GBM), anoligodendroglioma, or a medulloblastoma (including anaplastic andnon-anaplastic medulloblastoma). In other embodiments, the ALT-dependentcancer is an osteosarcoma.

Chemotherapeutic agent: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer. In one embodiment, achemotherapeutic agent is a radioactive compound. In one embodiment, achemotherapeutic agent is a biologic, such as a therapeutic monoclonalantibody. One of skill in the art can readily identify achemotherapeutic agent of use (see for example, Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 inAbeloff, Clinical Oncology 2^(nd) ed., © 2000 Churchill Livingstone,Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide toChemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D. S.,Knobf, M. F., Durivage, H. J. (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy isthe administration of more than one agent to treat cancer. One exampleis the administration of an oncolytic virus used in combination with aradioactive or chemical compound.

Deficient: As used herein, “ICP0-deficient” or “deficient in ICP0”refers to a recombinant virus having a disruption in the ICP0 gene (suchas one or more nucleotide insertions, deletions, point mutations, orcombinations thereof), which results in a substantial decrease in, orthe absence of, functional ICP0 protein. Similarly, “gC-deficient” or“deficient in gC” refers to a recombinant virus having a disruption inthe gC gene (such as one or more nucleotide insertions, deletions, pointmutations, or combinations thereof), which results in a substantialdecrease in, or the absence of, functional gC protein. The HSV-1 genomecontains two copies of the ICP0 gene and two copies of the gC gene,therefore an ICP0-deficient virus has a disruption of both copies of theICP0 gene, and a gC-deficient virus has a disruption of both copies ofthe gC gene. However, it is not necessary for both copies of the ICP0gene or the gC gene to have the same type of disruption. For example,one copy of the ICP0 (or gC) gene could be deleted (such as completelydeleted), while the second copy could contain a point mutation thatprevents expression of functional ICP0 (or gC) protein. Similarly, therecombinant viruses disclosed herein may be deficient in other genes,such as ICP34.5, ICP6 or ICP47, caused by a disruption in the gene. Asused herein, the term “diminishes or eliminates expression” offunctional protein (such as ICP0, gC, ICP34.5, ICP6 or ICP47 protein)does not refer to only a complete loss of expression, but also includesa decrease, in some cases a substantial decrease, in expression offunctional protein, such as a decrease of about 50%, about 60%, about70%, about 80%, about 90%, about 95%, about 98% or about 99%.

Disruption: As used herein, a “disruption” in a gene refers to anyinsertion, deletion or point mutation, or any combination thereof. Insome embodiments, the disruption leads to a partial or complete loss ofexpression of mRNA and/or functional protein.

Glycoprotein C (gC): An HSV attachment protein that mediates binding ofthe virus to cell surface heparan sulfate or chondroitin sulfate. gC isfurther described as Uniprot P10228. Viral entry of HSV involves viralenvelope proteins, glycoprotein C, glycoprotein B, which bind to heparansulfate. Glycoprotein D is also involved as viral entry and binds toother entry receptors. The HSV-1 gC protein is encoded by the UL44 gene.

Herpes simplex virus (HSV)-1: A member of the Alphaherpesvirinaesubfamily of the Herpesviridae family. Herpesviruses have a linear,double-stranded DNA genome that circularizes upon infection. The genomeis contained within an icosahedral capsid, which is surrounded by alipid envelope. The genome of HSV-1 is relatively complex and containstwo unique regions, called the long unique region (U_(L)) and the shortunique region (U_(S)), and includes two pairs of inverted repeatregions, TR_(L)/IR_(L) and IR_(S)/TR_(S) (see FIG. 8).

Heterologous: Originating from a separate genetic source or species.

Immunostimulatory molecule: Proteins that function to enhance an immuneresponse. Examples of immunostimulatory molecules include, but are notlimited to, granulocyte-macrophage colony-stimulating factor (GM-CSF),C-C motif chemokine ligand 5 (CCLS), C-C motif chemokine ligand 1(CCL1), interleukin (IL)-12 and B7.1.

Infected cell protein 0 (ICP0): An HSV-1 immediate early (IE)phosphoprotein that functions as a transactivator of viral and cellulargenes. ICP0 is important for the progression to lytic infection and forreactivation from latency. This protein includes a RING finger domainwith E3 ubiquitin ligase activity. ICP0 is also a minor structuralcomponent of the tegument layer of HSV-1 particles. The ICP0 gene ispresent in two copies within the HSV-1 genome, one copy in each of theTR_(L) and IR_(L) inverted repeat regions. ICP0 is further described asUniProt P08393. The amino acid sequence of ICP0 is found in GenBankAccession No. AEQ77030.1.

Infected cell protein 6 (ICP6): A protein encoded by HSV-1 thatfunctions as a ribonucleoside-diphosphate reductase holoenzyme. ICP6,which is also known as ribonucleoside-diphosphate reductase largesubunit, is encoded by the UL39 gene. In the context of the presentdisclosure, the term “ICP6 gene” is used to refer to the gene encodingthe ICP6 protein. ICP6 is further described as UniProt P08543.

Infected cell protein 34.5 (ICP34.5): A protein encoded by HSV-1 thatcontributes to HSV resistance to the antiviral effects of α/βinterferon. ICP34.5 also down-regulates host MHC class II proteins andacts as a neurovirulence factor. ICP34.5 is encoded by the RL1 gene. Inthe context of the present disclosure, the term “ICP34.5 gene” is usedto refer to the gene encoding the ICP34.5 protein. ICP34.5 is furtherdescribed as UniProt P36313.

Infected cell protein 47 (ICP47): A protein encoded by HSV-1 thatinhibits the MHC class I pathway in host cells by preventing binding ofantigen to TAP. ICP47 is encoded by the

US12 gene. In the context of the present disclosure, the term “ICP47gene” is used to refer to the gene encoding the ICP47 protein. ICP47 isfurther described as UniProt P03170.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or organelle, has been substantiallyseparated or purified away from other biological components in theenvironment (such as a cell) in which the component naturally occurs,i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins andorganelles. Nucleic acids and proteins that have been “isolated” includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Modification: A change in the sequence of a nucleic acid or proteinsequence. For example, amino acid sequence modifications include, forexample, substitutions, insertions and deletions, or combinationsthereof. Insertions include amino and/or carboxyl terminal fusions aswell as intrasequence insertions of single or multiple amino acidresidues. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. In some embodimentsherein, the modification (such as a substitution, insertion or deletion)results in a change in function, such as a reduction or enhancement of aparticular activity of a protein. As used herein, “Δ” or “delta” referto a deletion. Substitutional modifications are those in which at leastone residue has been removed and a different residue inserted in itsplace. Amino acid substitutions are typically of single residues, butcan occur at a number of different locations at once. Substitutions,deletions, insertions or any combination thereof may be combined toarrive at a final mutant sequence. These modifications can be preparedby modification of nucleotides in the DNA encoding the protein, therebyproducing DNA encoding the modification. Techniques for makinginsertion, deletion and substitution mutations at predetermined sites inDNA having a known sequence are well known in the art. A “modified”protein, nucleic acid or virus is one that has one or more modificationsas outlined above.

Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growthof tissue or cells that results from excessive cell division. Neoplasticgrowth can produce a tumor. The amount of a tumor in an individual isthe “tumor burden” which can be measured as the number, volume, orweight of the tumor. A tumor that does not metastasize is referred to as“benign.” A tumor that invades the surrounding tissue and/or canmetastasize is referred to as “malignant.”

Oncolytic virus: A virus that selectively kills cells of a proliferativedisorder, e.g., cancer/tumor cells. Killing of the cancer cells can bedetected by any method established in the art, such as determiningviable cell count, or detecting cytopathic effect, apoptosis, orsynthesis of viral proteins in the cancer cells (e.g., by metaboliclabeling, immunoblot, or RT-PCR of viral genes necessary forreplication), or reduction in size of a tumor.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the therapeutic recombinant viruses disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop,such as a reduction in tumor burden (such as the volume or size of atumor) or a decrease in the number of size of metastases. “Ameliorating”refers to the reduction in the number or severity of signs or symptomsof a disease, such as cancer.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Inone embodiment, a preparation is purified such that the protein orpeptide represents at least 50% of the total peptide or protein contentof the preparation. Substantial purification denotes purification fromother proteins or cellular components. A substantially purified proteinis at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific,non-limiting example, a substantially purified protein is 90% free ofother proteins or cellular components.

Recombinant: A recombinant nucleic acid molecule, protein or virus isone that has a sequence that is not naturally occurring or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination can beaccomplished by chemical synthesis or by the artificial manipulation ofisolated segments of nucleic acid molecules, such as by geneticengineering techniques. The term “recombinant” also includes nucleicacids, proteins and viruses that have been altered solely by addition,substitution, or deletion of a portion of the natural nucleic acidmolecule, protein or virus.

Sequence identity: The similarity between amino acid or nucleic acidsequences is expressed in terms of the similarity between the sequences,otherwise referred to as sequence identity. Sequence identity isfrequently measured in terms of percentage identity (or similarity orhomology); the higher the percentage, the more similar the two sequencesare. Homologs or variants of a polypeptide or nucleic acid molecule willpossess a relatively high degree of sequence identity when aligned usingstandard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of an antibody that specifically binds a targetantigen or a fragment thereof are typically characterized by possessionof at least about 75%, for example at least about 80%, 90%, 95%, 96%,97%, 98% or 99% sequence identity counted over the full length alignmentwith the amino acid sequence of the antibody using the NCBI Blast 2.0,gapped blastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

Synthetic: Produced by artificial means in a laboratory, for example asynthetic nucleic acid or protein can be chemically synthesized in alaboratory.

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthor metastasis of a tumor. In one embodiment, a therapeutically effectiveamount is the amount necessary to eliminate, reduce the size, or preventmetastasis of a tumor. When administered to a subject, a dosage willgenerally be used that will achieve target tissue concentrations (forexample, in tumors) that has been shown to achieve a desired in vitroeffect.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. “Comprising A or B” means including A, or B, or Aand B. It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Introduction

Cellular immortality is a hallmark of cancer that distinguishes tumorcells from normal cells. When a normal somatic cell divides, telomericDNA at the ends of its chromosomes becomes shorter. If telomeres shortentoo much, an alarm signal is generated, and the cell permanently stopsdividing and enters senescence or undergoes apoptosis. Telomereshortening therefore acts as a biological mechanism for limitingcellular life span. Cancer cells, on the other hand, can becomeimmortalized by activating a telomere maintenance mechanism (TMM) thatcounteracts telomere shortening by synthesizing new telomeric DNA fromeither an RNA template using the enzyme telomerase or a DNA templateusing a mechanism called alternative lengthening of telomeres (ALT).Because the presence of a TMM is a nearly universal characteristic ofcancer cells, and repressing these mechanisms results in cancer cellsenescence or death, TMMs have become targets for treating cancer.However, there are currently no ALT-targeted cancer therapies.

It is disclosed herein that the generation of extrachromosomal telomericrepeat (ECTR) DNA in ALT resembles HSV-1 viral genomes and triggers asimilar protective response that prevents both viral and tumorreplication. This novel anti-viral/anti-tumor DNA immune responsesignature is referred to herein as the “Viral and Extrachromosomal DNATranscriptional Response (VECTR).” VECTR triggers immune effectors thatprotect the host from extrachromosomal viral and cellular DNA. It isfurther disclosed herein that the VECTR response is inactivatedspecifically in ALT-dependent, but not telomerase-dependent cancers.This finding led to the prediction that ICP0-null, and/or glycoprotein C(gC) deficient HSV-1 viruses would replicate specifically in cancercells, but leave normal cells unharmed. To test this, wild-type, gCdeficient and ICP0-null HSV-1 virus replication and killing werecompared in a panel of telomerase-positive tumors, ALT tumors and normalcells. The results demonstrated that HSV-1 ICP0-null, gC deficientviruses replicate specifically in ALT-dependent cancers. The studiesdisclosed herein indicate that HSV-1 ICP0-null and/or gC deficientviruses can be used as a selective therapy for treating ALT-dependentcancers.

IV. Overview of Several Embodiments

Provided herein are methods of treating an ALT-dependent cancer in asubject. The method includes administering to the subject a recombinant,ICP0-deficient HSV-1. In some embodiments, the method further includesselecting a subject having an ALT-dependent cancer. In some embodiments,the ICP0-deficient HSV-1 has a disruption in the ICP0 gene thatdiminishes or eliminates expression of functional ICP0. In someexamples, the disruption in the ICP0 gene includes a complete deletionof the ICP0 gene, a partial deletion of the ICP0 gene, an insertion inthe ICP0 gene, a point mutation in the ICP0 gene, or any combinationthereof. The HSV-1 genome includes two copies of the ICP0 gene, a firstcopy in TR_(L) and a second copy in IR_(L). In the context of thepresent disclosure, an ICP0-deficient virus has a disruption of bothcopies of the ICP0 gene. However, it is not necessary for both copies ofthe ICP0 gene to have the same type of disruption. As one non-limitingexample, one copy of the ICP0 gene can be completely deleted, while thesecond copy can contain a point mutation, partial deletion or insertionthat prevents expression of functional ICP0 protein.

In some embodiments, the recombinant ICP0-deficient HSV-1 includes acomplete deletion of the ICP0 gene. In particular examples, therecombinant HSV-1 includes a complete deletion of both copies of theICP0 gene.

In some embodiments, the recombinant ICP0-deficient HSV-1 includes apartial deletion in the ICP0 gene. In particular examples, the partialdeletion in the ICP0 gene is in the RING finger domain coding region.

In some embodiments, the recombinant ICP0-deficient HSV-1 includes aninsertion or a point mutation in the ICP0 gene. In particular examples,the insertion or point mutation in the ICP0 gene is in the RING fingerdomain coding region.

In some embodiments, the partial deletion, insertion or point mutationin the ICP0 gene diminishes or eliminates E3 ubiquitin ligase activityof ICP0.

Nonsense mutations at codon 212, 428, 525, and 680 of the ICP0 gene ofHSV-1 result in a replication-defective virus that is rescued by U2OScells. Therefore, mutations in the ICP0 gene up to codon 680 that resultin a truncated, functionally inactive, ICP0 protein are contemplatedherein. In addition, the RING finger domain within ICP0 has been shownto be important for ICP0 function. Therefore, mutations that disrupt thefunction of the RING finger domain, thereby resulting in functionalinactivation of ICP0, are further contemplated. One such ICP0 mutationthat has been previously described is the FxE mutant (Everett et al., JGen Virol 70:1185-1202, 1989; Everett et al., J Virol 78(4):1763-1774,2004).

In some embodiments, the recombinant HSV-1 is glycoprotein C (gC)deficient. In a particular example, the recombinant HSV-1 has a gC genewith a deletion of the cytosine at position 186 relative to the gC geneof wild-type HSV-1. In a particular example, the recombinant HSV-1includes a gC gene with a premature termination at the 175^(th) codon ofGc relative to the gC gene of wild-type HSV-1. In a particular example,the recombinant HSV-1 includes the mutations shown in FIG. 10, inassociation with HSV-1 strain d11403. These mutations include a deletionof the cytosine at position 186, and a premature termination at codon175 (as described in Cunha, et al. Widely used Herpes Simplex Virus 1ICP0 Deletion Mutant Strain d11403 and its Derivative Viruses Do NotExpress Glycoprotein C Due to a Secondary Mutation in the gC gene. PLOSone. Jul. 17, 2015., incorporated by reference herein). In someembodiments, the HSV-1 has a modification of the gC gene that diminishesor eliminates expression of gC, or that results in expression of atruncated gC.

In some embodiments, the recombinant HSV-1 is both ICP0-deficient and gCdeficient.

In some embodiments, the recombinant HSV-1 (which can be ICP0-deficient,gC deficient, or both) includes a heterologous gene. In some examples,the heterologous gene encodes an immunostimulatory molecule. Inparticular examples, the immunostimulatory molecule isgranulocyte-macrophage colony-stimulating factor (GM-CSF), C-C motifchemokine ligand 5 (CCLS), C-C motif chemokine ligand 1 (CCL1),interleukin (IL)-12 or B7.1.

In some embodiments, the recombinant ICP0-deficient HSV-1 furtherincludes a disruption in one or more additional HSV-1 genes thatdiminishes or eliminates expression of functional protein encoded by thegene(s). In some examples, the recombinant virus further includes adisruption in the ICP47 gene. In some examples, recombinant virusfurther includes a disruption in the ICP6 gene. In some examples,recombinant virus further includes a disruption in the ICP34.5 gene. Thedisruption in the ICP47, ICP6 and/or ICP34.5 genes can be a completedeletion, a partial deletion, a point mutation, an insertion, or anycombination thereof.

In particular non-limiting embodiments, the recombinant ICP0-deficientHSV-1 includes a complete deletion of the ICP0 gene and includes aheterologous gene encoding GM-CSF. In some examples, the recombinantHSV-1 has a genome sequence at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 15. In specific examples, the genome sequence ofthe recombinant HSV-1 comprises or consists of SEQ ID NO: 15.

In other particular non-limiting embodiments, the recombinantICP0-deficient HSV-1 includes a complete deletion of the ICP0 gene, apartial deletion of the ICP47 gene, and includes a heterologous geneencoding GM-CSF. In some examples, the recombinant HSV-1 has a genomesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 16. In specific examples, the genome sequence of the recombinantHSV-1 comprises or consists of SEQ ID NO: 16.

In particular non-limiting embodiments, the recombinant ICP0-deficientHSV-1 includes a complete deletion of the ICP0 gene, a mutation in thegC gene (such that it is gC deficient), and includes a heterologous geneencoding GM-CSF. In some examples, the recombinant HSV-1 has a genomesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO: 15. In specific examples, the genome sequence of the recombinantHSV-1 comprises or consists of SEQ ID NO: 23.

In other particular non-limiting embodiments, the recombinantICP0-deficient HSV-1 includes a complete deletion of the ICP0 gene, apartial deletion of the ICP47 gene, a mutation of the gC gene (such thatit is gC deficient), and includes a heterologous gene encoding GM-CSF.In some examples, the recombinant HSV-1 has a genome sequence at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to SEQ ID NO: 16. Inspecific examples, the genome sequence of the recombinant HSV-1comprises or consists of SEQ ID NO: 24.

Also provided is a method of selectively killing an ALT-dependent tumorcell by administering a recombinant HSV disclosed herein (such as onethat is ICP0-deficient, gC deficient, or both ICP0-deficient and gCdeficient). In some embodiments, the method is an in vitro method thatincludes contacting an ALT-dependent tumor cell with a recombinantoncolytic HSV-1. In other embodiments, the method is an in vivo methodthat includes administering an oncolytic HSV-1 disclosed herein to asubject having an ALT-dependent cancer. In some examples, the in vivomethod further includes the step of selecting a subject having anALT-dependent cancer.

In some embodiments of the methods disclosed herein, the method furtherincludes administering an anti-cancer therapy to the subject. Anappropriate anti-cancer therapy can be selected by a medicalpractitioner. In some examples, the anti-cancer therapy comprisesadministration of a telomerase inhibitor, chemotherapy, or radiationtherapy. In other examples, the anti-cancer therapy includes surgery,such as surgical resection of the tumor.

In specific examples, the recombinant HSV-1 (such as one that isICP0-deficient, gC deficient, or both ICP0-deficient and gC deficient)is used in combination with a telomerase inhibitor. Telomeraseinhibitors have the potential to treat cancers that use telomerase tomaintain telomeres. Resistance occurs through the down-regulation oftelomerase and the switch to ALT to maintain telomere length. Treatingtumors with a telomerase inhibitor and an ICP0 deleted HSV-1 inhibitsboth means by which a cancer cell can maintain telomere length.Telomerase inhibitors include, for example, inhibitors that target thetelomerase reverse transcriptase (TERT) catalytic subunit) or inhibitorsthat target telomerase RNA. TERT inhibitors include, but are not limitedto, antisense oligonucleotides, small interfering RNA (siRNA) anddouble-stranded RNA (dsRNA) that reduce TERT mRNA, and small moleculessuch as 3′-azido-2′,3′-dideoxythymine (AZT) and BIBR1532. Examples oftelomerase RNA inhibitors include antisense oligonucleotides, hammerheadribozymes and siRNA (Andrews and Tollefsbol, Methods Mol Biol 405:1-8,2008).

In some embodiments, the ALT-dependent cancer is a cancer that isresistant to telomerase inhibitors and/or TERT inhibitors.

In some embodiments herein, the ALT-dependent cancer is a soft tissuesarcoma, such as but not limited to, a pleomorphic sarcoma, afibrosarcoma, a leiomyosarcoma, a liposarcoma, an angiosarcoma, anepithelioid sarcoma or a chondrosarcoma. In other embodiments, theALT-dependent cancer is a cancer of the central nervous system, such asbut not limited to an astrocytoma (for example, a diffuse astrocytoma oran anaplastic astrocytoma), GBM, an oligodendroglioma, or amedulloblastoma (including anaplastic and non-anaplasticmedulloblastoma). In other embodiments, the ALT-dependent cancer is anosteosarcoma.

In order to identify an ALT-dependent cancer, and thereby select asubject having an ALT-dependent cancer who would be responsive to HSVoncolytic therapy, a biopsy taken from the subject's tumor can bescreened for one or more hallmarks or indicators of ALT. ALT generatesextrachromasomal telomeric repeat (ECTR) DNA that associates andco-localizes with PML nuclear bodies to form ALT-associated PML bodies(APBs). Fluorescence in situ hybridization for telomeric rich DNAtogether with immunofluorescence for PML NBs for the detection of APBscan be used as a marker for ALT. ECTR DNA can be linear double-stranded,circular double-stranded or circular partially double-stranded(C-Circle). Detection of C-circles via a C-circle assay can also be usedto determine whether a tumor cell uses ALT to maintain chromosomes. Thelevel of C-circle DNA in cancer cells has been shown to accuratelyreflect the level of ALT activity, and this biomarker can be found inthe blood of patients who have ALT-dependent cancers (Henson et al., NatBiotechnol 27:1181-1185, 2009). In addition, mutations in ATRx and H3.3have been reported to associate with ALT and could also be used as anindicator of an ALT-dependent cancer. These assays can be usedindividually or in combination with the measurement of telomerase levelsor telomerase activity to increase the accuracy of identifying ALTcancers. In addition, it is demonstrated herein that the introduction ofextrachromosomal DNA, such as a plasmid or sheared calf thymus DNA, intoa telomerase-positive cell induces the expression of IFIT1, IFIT2,IFIT3, OAS1 and OAS2, while an ALT cell fails to elicit a similarresponse. This differential response can also be used in order toidentify ALT-dependent cancers.

Further provided herein are recombinant ICP0-deficient HSV-1 thatinclude a heterologous gene. In some embodiments, the heterologous geneis an immunostimulatory molecule. In particular examples, theimmunostimulatory molecule is GM-CSF, CCLS, CCL1, IL-12 or B7.1

In particular examples, the recombinant HSV-1 includes a completedeletion of the ICP0 gene and a heterologous gene encoding GM-CSF. Inspecific examples, the recombinant HSV-1 has a genome sequence at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to SEQ ID NO: 15. In onenon-limiting example, the genome sequence of the recombinant HSV-1comprises or consists of SEQ ID NO: 15.

Also provided herein are recombinant ICP0-deficient HSV-1 that includedisruptions in one or more additional HSV-1 genes. In some embodiments,the one or more genes are selected from ICP34.5, ICP6 and ICP47. In someexamples, the disruption is a complete deletion, a partial deletion, apoint mutation or an insertion. In some instances, the recombinant HSV-1further includes a heterologous gene, such as, but not limited to, agene encoding an immunostimulatory molecule.

In particular examples, the recombinant HSV-1 includes a completedeletion of the ICP0 gene, a partial deletion of the ICP47 gene and aheterologous gene encoding GM-CSF. In specific examples, the recombinantHSV-1 has a genome sequence at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 16. In one non-limiting example, the genomesequence of the recombinant HSV-1 comprises or consists of SEQ ID NO:16.

Use of the recombinant HSV-1 disclosed herein methods of treating anALT-dependent cancer in a subject is also provided herein. The methodincludes administering a recombinant HSV-1 disclosed herein to thesubject. In some embodiments, the method further includes selecting asubject having an ALT-dependent cancer.

V. Pharmaceutical Compositions of Oncolytic HSV

Provided herein are compositions comprising a recombinant HSV, such asone that is ICP0-deficient, gC deficient, or both ICP0-deficient and gCdeficient. The compositions are, optionally, suitable for formulationand administration in vitro or in vivo. Optionally, the compositionscomprise one or more of the provided agents and a pharmaceuticallyacceptable carrier. Suitable carriers and their formulations aredescribed in Remington: The Science and Practice of Pharmacy, 22^(nd)Edition, Loyd V. Allen et al., editors, Pharmaceutical Press (2012).Pharmaceutically acceptable carriers include materials that are notbiologically or otherwise undesirable, i.e., the material isadministered to a subject without causing undesirable biological effectsor interacting in a deleterious manner with the other components of thepharmaceutical composition in which it is contained. If administered toa subject, the carrier is optionally selected to minimize degradation ofthe active ingredient and to minimize adverse side effects in thesubject.

The recombinant viruses are administered in accord with known methods,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, intratumoral or inhalation routes. Theadministration may be local (such as intratumoral) or systemic. Thecompositions can be administered via any of several routes ofadministration, including topically, orally, parenterally,intravenously, intra-articularly, intraperitoneally, intramuscularly,subcutaneously, intracavity, transdermally, intrahepatically,intracranially, nebulization/inhalation, or by installation viabronchoscopy. Thus, the compositions are administered in a number ofways depending on whether local or systemic treatment is desired, and onthe area to be treated.

In some embodiments, the compositions for administration will include arecombinant virus as described herein dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of active agent in these formulations can vary widely, andwill be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the subject's needs.

Pharmaceutical formulations, particularly, of the recombinant virusescan be prepared by mixing the recombinant virus having the desireddegree of purity with optional pharmaceutically acceptable carriers,excipients or stabilizers. Such formulations can be lyophilizedformulations or aqueous solutions.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations used. Acceptable carriers,excipients or stabilizers can be acetate, phosphate, citrate, and otherorganic acids; antioxidants (e.g., ascorbic acid) preservatives, lowmolecular weight polypeptides; proteins, such as serum albumin orgelatin, or hydrophilic polymers such as polyvinylpyllolidone; and aminoacids, monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents; and ionic and non-ionicsurfactants (e.g., polysorbate); salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants. The recombinant HSV can be formulated at any appropriateconcentration of infectious units.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the recombinant virussuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The recombinant HSV, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the provided methods, compositions can beadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically intratumorally, or intrathecally. Insome embodiments, parenteral administration, intratumoraladministration, or intravenous administration are the methods ofadministration. The formulations of compounds can be presented inunit-dose or multi-dose sealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced or infected by recombinant HSV or transfected with nucleicacids for ex vivo therapy can also be administered intravenously orparenterally as described above.

In some embodiments, the pharmaceutical preparation is in unit dosageform. In such form the preparation is subdivided into unit dosescontaining appropriate quantities of the active component. Thus, thepharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include, but are notlimited to, powder, tablets, pills, capsules and lozenges.

V. Methods of Treatment Using Oncolytic HSV

The recombinant HSV compositions disclosed herein (such as those thatinclude an HSV that is ICP0-deficient, gC deficient, or bothICP0-deficient and gC deficient) can be administered for therapeutic orprophylactic treatment. In particular, provided are methods ofinhibiting ALT-dependent tumor cell viability in a subject, inhibitingALT-dependent tumor progression in a subject, reducing ALT-dependenttumor volume in a subject and/or treating ALT-dependent cancer in asubject. The methods include administering a therapeutically effectiveamount of a recombinant HSV (or composition thereof) to the subject. Insome embodiments, the method further includes selecting a subject havingan ALT-dependent cancer. As described throughout, the oncolytic HSV orpharmaceutical composition is administered in any number of waysincluding, but not limited to, intravenously, intravascularly,intrathecally, intramuscularly, subcutaneously, intratumorally,intraperitoneally, or orally. Optionally, the method further comprisesadministering to the subject one or more additional therapeutic agents.In some embodiments, the therapeutic agent is a chemotherapeutic agent(such as cisplatin, 5-FU, carboplatin, and the like). In someembodiments, the therapeutic agent is a biologic agent (such astrastuzumab, cetuximab, ribuximab, panitumumab, and the like, see forexample Scott et al., Nature Reviews Cancer 12:278-287, 2012, hereinincorporated by reference). In other embodiments, the therapeutic agentis a telomerase inhibitor. In other embodiments, the therapeutic agentis an immune modulator.

In some embodiments, the ALT-dependent cancer or tumor is a soft tissuesarcoma, an osteosarcoma, or a cancer of the central nervous system,such as an astrocytoma. In some cases, the cancer is metastatic. In someexamples, the tumor is a tumor of the mammary, pituitary, thyroid, orprostate gland; a tumor of the brain, liver, meninges, bone, ovary,uterus, or cervix; monocytic or myelogenous leukemia; adenocarcinoma,adenoma, astrocytoma, bladder tumor, brain tumor, Burkitt's lymphoma,breast carcinoma, cervical carcinoma, colon carcinoma, kidney carcinoma,liver carcinoma, lung carcinoma, ovarian carcinoma, pancreaticcarcinoma, prostate carcinoma, rectal carcinoma, skin carcinoma, stomachcarcinoma, testis carcinoma, thyroid carcinoma, chondrosarcoma,choriocarcinoma, fibroma, fibrosarcoma, glioblastoma, glioma, hepatoma,histiocytoma, leiomyoblastoma, leiomyosarcoma, lymphoma, liposarcomacell, mammary tumor, medulloblastoma, myeloma, plasmacytoma,neuroblastoma, neuroglioma, osteogenic sarcoma, pancreatic tumor,pituitary tumor, retinoblastoma, rhabdomyosarcoma, sarcoma, testiculartumor, thymoma, or Wilms tumor. Tumors include both primary andmetastatic solid tumors, including carcinomas of breast, colon, rectum,lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver,gallbladder and bile ducts, small intestine, urinary tract (includingkidney, bladder and urothelium), female genital tract, (includingcervix, uterus, and ovaries as well as choriocarcinoma and gestationaltrophoblastic disease), male genital tract (including prostate, seminalvesicles, testes and germ cell tumors), endocrine glands (including thethyroid, adrenal, and pituitary glands), and skin, as well ashemangiomas, melanomas, sarcomas (including those arising from bone andsoft tissues as well as Kaposi's sarcoma) and tumors of the brain,nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas). In some aspects, solid tumors may be treated thatarise from hematopoietic malignancies such as leukemias (i.e. chloromas,plasmacytomas and the plaques and tumors of mycosis fungoides andcutaneous T-cell lymphoma/leukemia) as well as in the treatment oflymphomas (both Hodgkin's and non-Hodgkin's lymphomas). In addition,treatments may be useful in the prevention of metastases from the tumorsdescribed herein.

In therapeutic applications, recombinant HSV or compositions thereof areadministered to a subject in a therapeutically effective amount or dose.Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health. Single ormultiple administrations of the compositions may be administereddepending on the dosage and frequency as required and tolerated by thepatient. A “patient” or “subject” includes both humans and otheranimals, particularly mammals (such as cats, dogs, cows, sheep, andpigs). Thus, the methods are applicable to both human therapy andveterinary applications.

An effective amount of a recombinant HSV is determined on an individualbasis and is based, at least in part, on the particular recombinantvirus used; the individual's size, age, gender; and the size and othercharacteristics of the proliferating cells. For example, for treatmentof a human, at least 10³ plaque forming units (PFU) of a recombinantvirus is used, such as at least 10⁴, at least 10⁵, at least 10⁶, atleast 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰, at least 10¹¹, orat least 10¹² PFU, for example approximately 10³ to 10¹² PFU of arecombinant virus is used, depending on the type, size and number ofproliferating cells or neoplasms present. The effective amount can befrom about 1.0 pfu/kg body weight to about 10¹⁵ pfu/kg body weight(e.g., from about 10² pfu/kg body weight to about 10¹³ pfu/kg bodyweight). A recombinant HSV is administered in a single dose or inmultiple doses (e.g., two, three, four, six, or more doses). Multipledoses can be administered concurrently or consecutively (e.g., over aperiod of days or weeks).

Administration of the HSV-1 oncolytic viruses disclosed herein can alsobe accompanied by administration of other anti-cancer agents ortherapeutic treatments (such as surgical resection of a tumor and/ortreatment with a telomerase inhibitor). Any suitable anti-cancer agentcan be administered in combination with the recombinant virusesdisclosed herein. Exemplary anti-cancer agents include, but are notlimited to, chemotherapeutic agents, such as, for example, mitoticinhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, anti-survival agents, biological responsemodifiers, anti-hormones (e.g. anti-androgens), CDK inhibitors andanti-angiogensesis agents. Other anti-cancer treatments includeradiation therapy and other antibodies that specifically target cancercells (e.g., biologics).

Non-limiting examples of alkylating agents include nitrogen mustards(such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).

Non-limiting examples of antimetabolites include folic acid analogs(such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine),and purine analogs, such as mercaptopurine or thioguanine.

Non-limiting examples of natural products include vinca alkaloids (suchas vinblastine, vincristine, or vindesine), epipodophyllotoxins (such asetoposide or teniposide), antibiotics (such as dactinomycin,daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), andenzymes (such as L-asparaginase).

Non-limiting examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocroticalsuppressants (such as mitotane and aminoglutethimide).

Non-limiting examples of hormones and antagonists includeadrenocorticosteroids (such as prednisone), progestins (such ashydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrolacetate), estrogens (such as diethylstilbestrol and ethinyl estradiol),antiestrogens (such as tamoxifen), and androgens (such as testeroneproprionate and fluoxymesterone). Examples of the most commonly usedchemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan,CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin),Xeloda (Capecitabine), Zevelin and calcitriol.

Non-limiting examples of immunomodulators that can be used includeAS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon(Genentech), GM-CSF (granulocyte macrophage colony stimulating factor;Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immuneglobulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.),SK&F 106528, and TNF (tumor necrosis factor; Genentech).

Another common treatment for some types of cancer is surgical treatment,for example surgical resection of the cancer or a portion of it. Anotherexample of a treatment is radiotherapy, for example administration ofradioactive material or energy (such as external beam therapy) to thetumor site to help eradicate the tumor or shrink it prior to surgicalresection.

CDK (Cyclin-dependent kinase) inhibitors are agents that inhibit thefunction of CDKs. Non-limiting examples of CDK inhibitors for use in theprovided methods include AG-024322, AT7519, AZD5438, flavopiridol,indisulam, P1446A-05, PD-0332991, and P276-00 (see e.g., Lapenna et al.,Nature Reviews, 8:547-566 , 2009). Other CDK inhibitors includeLY2835219, Palbociclib, LEE011 (Novartis), pan-CDK inhibitor AT7519,seliciclib, CYC065, butyrolactone I, hymenialdisine, SU9516, CINK4,PD0183812 or fascaplysin.

In some examples, the CDK inhibitor is a broad-range inhibitor (such asflavopiridol, olomoucine, roscovitine, kenpaullone, SNS-032, AT7519,AG-024322, (S)-Roscovitine or R547). In other examples, the CDKinhibitor is a specific inhibitor (such as fascaplysin, ryuvidine,purvalanol A, NU2058, BML-259, SU 9516, PD0332991 or P-276-00).

The choice of agent and dosage can be determined readily by one of skillin the art based on the given disease being treated. Combinations ofagents or compositions can be administered either concomitantly (e.g.,as a mixture), separately but simultaneously (e.g., via separateintravenous lines) or sequentially (e.g., one agent is administeredfirst followed by administration of the second agent). Thus, the termcombination is used to refer to concomitant, simultaneous or sequentialadministration of two or more agents or compositions.

According to the methods disclosed herein, the subject is administeredan effective amount of one or more of the agents provided herein. Theeffective amount is defined as any amount necessary to produce a desiredphysiologic response (e.g., killing of an ALT-dependent cancer cell).Therapeutic agents are typically administered at the initial dosage ofabout 0.001 mg/kg to about 1000 mg/kg daily. A dose range of about 0.01mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, orabout 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg,can be used. The dosages, however, may be varied depending upon therequirements of the subject, the severity of the condition beingtreated, and the compound being employed. For example, dosages can beempirically determined considering the type and stage of cancerdiagnosed in a particular subject. The dose administered to a subject,in the context of the provided methods should be sufficient to affect abeneficial therapeutic response in the patient over time. Determinationof the proper dosage for a particular situation is within the skill ofthe practitioner. Thus, effective amounts and schedules foradministering the agent may be determined empirically by one skilled inthe art. The dosage should not be so large as to cause substantialadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. The dosage can be adjusted by the individualphysician in the event of any contraindications. Guidance can be foundin the literature for appropriate dosages for given classes ofpharmaceutical products.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Materials and Methods

This examples describes the materials and experimental procedures usedfor the studies described in Example 2.

Cell Lines

HOS, SJSA1, U2OS, SAOS2, KMST6, SUSM-1, SKBR3, SK-LU-1, HeLa LT(O'Sullivan et al., Nat Struct Mol Biol 21, 167-174, 2014), HFF, DAOY,A549, H1299, L229N, A172, U87MG, T98G, DBTRG, WI-38 VA13/R2 GM847,SJRH30, and G292 were grown in DMEM with 10% FBS at 37° C. and 5% CO₂.BJ cells were grown in MEM, NEAA, sodium pyruvate and 10% FBS at 37° C.and 5% CO₂. SAEC were from Lonza and grown in SABM supplemented withSAGM SINGLEQUOTS™ and grown at 37° C. in 5% CO₂ and 3% O₂. LS2, HIO107,HIO107 hTERT, HIO118 and HIO 118 hTERT cells were provided by DomoniqueBroccoli (Plantinga et al., Mol Cancer Res 11, 557-567, 2013; Mitchell,Mol Cancer Ther 9, 682-692, 2010). LS2 cells were grown in RPMIsupplemented with 1× GLUTAMAX™ (Life Technologies), 1× MEM essentialvitamin mix (Lonza cat # 13-607c), 1× ITES (Lonza cat # 17-839z), 1×Pen/Strep/L-glut (Life Technologies cat # 10378-016), 1 mM sodiumpyruvate (MP Biomedical cat # 1682049), 1× non-essential amino acids(Lonza cat # 13-114E) and 20% FBS at 37° C. and 5% CO₂. HIO cells weregrown in a 1:1 dilution of Medium 199 (Sigma-Aldrich cat # M4530) andMCDB 105 (Sigma cat # M6395) supplemented with 4% FBS, 1× Glutagro(Cellgro cat # 25-015-C1) and 0.2 U insulin (Santa Cruz Biotechnologycat # sc-360248) at 37° C. and 5% CO₂. SW872 were purchased from ATCCand initially cultured in L15 medium supplemented with 10% FBS at 37° C.without CO₂. These cells were then conditioned to grow in LS2 medium andall subsequent experiments with these cells were done in LS2 medium.

Viruses, Infections and Plaque Assay

The HSV-1 strains used in the studies disclosed herein were the 17syn+wild type (WT) and the matched ICP0 deletion mutant (ΔICP0) d11403 (Stowand Stow, J Gen Virol 67 (Pt 12), 2571-2585, 1986). Viruses were grownin Vero cells as follows. A 90% confluent 15 cm plate was infected at anMOI of 0.001 for HSV-1 WT and 0.1 for HSV-1 ΔICP0 and incubated untilcells were rounded but still attached to the plate, typically 3-5 days.Both the media and cells were collected and slowly frozen at −80° C.followed by a quick thaw at 37° C. for a total of 3 freeze/thaw cycles.The mixture was spun at 3000 RPM for 15 minutes and the resultingsupernatant was collected and spun for an additional 15 minutes at 3000RPM to remove any residual cell debris. The supernatant from this spinwas collected, aliquoted and stored at −80° C.

For tittering, one aliquot was thawed and tittered on U2OS cells byplaque assay since these cells fully rescue replication of ICP0 deletedHSV-1. U2OS cells (100,000) were plated in 24-well plates and allowed toattach for 6 hours. The virus was serial diluted in DMEM without serumyielding a dilution range from 10⁻³ to 10⁻⁸. The diluted virus was addedto the cells (3 wells/dilution) and incubated for 1 hour at 37° C. Thevirus was removed and replaced with DMEM containing 10% FBS and 1% humanserum and incubated for 36-48 hours until clear plaques were visible. Tobetter visualize the plaques, the media was removed and 1 mg/mL crystalviolet dissolved in 20% ethanol was added to each well and incubated for10 minutes. Each well was washed once with PBS and plaques were countedin the well with the least number of observable plaques. The averagenumber of plaques from 3 wells of the same dilution was calculated. Thetiter equals the average number of plaquescounted×10^((dilution number)) pfu/mL. For all other plaque assays, thecells were infected at the indicated multiplicity of infection (MOI),which was calculated based on viral titer and number of cells per well.

To examine the co-localization of incoming HSV-1 viral genomes with PMLNBs, cells were infected with HSV-1 WT or ΔICP0 as described above andincubated for 24 hours. Cells were washed, fixed and stained withantibodies against ICP4 and PML. The edges of plaques were imaged byconfocal microscopy at 63x magnification for cells in the early stagesof infection.

RT-qPCR analysis

The Ambion PURELINK™ RNA Mini Kit (cat # 12183018A) was used for theisolation of RNA according to the manufacturer's recommendations. TotalRNA was DNase treated using the Ambion DNA-free kit (cat # AM1906) and 1μg of total RNA was reverse transcribed using the Bio-Rad iScriptReverse Transcription Supermix for RT-qPCR (cat # 170-8840). The cDNAwas diluted to a final volume of 100 μL and 2 μL was used to set up 10μL RT-qPCR reactions with Kapa SYBR™ Fast qPCR Universal Master Mix(Kapa Biosystems cat # KK4601). All samples were run in duplicate on aBio-Rad CFX96 real-time system. The reactions were heated to 95° C. for30 seconds followed by 40 cycles of 95° C. for 5 seconds and 61° C. for30 seconds. Following the 40 cycles, a melt curve analysis was performedto ensure amplification of a single product. The target genes weredetected using the following primers:

IFIT1 (Fwd) (SEQ ID NO: 1) TGTCCAAGGTGGTAAAGGGTG IFIT1 (Rev)(SEQ ID NO: 2) CAGGTCACCAGACTCCTCAC IFIT2 (Fwd) (SEQ ID NO: 3)AAGCACCTCAAAGGGCAAAAC IFIT2 (Rev) (SEQ ID NO: 4) TCGGCCCATGTGATAGTAGACIFIT3 (Fwd) (SEQ ID NO: 5) TCAGAAGTCTAGTCACTTGGGG IFIT3 (Rev)(SEQ ID NO: 6) ACACCTTCGCCCTTTCATTTC OAS1 (Fwd) (SEQ ID NO: 7)TGTCCAAGGTGGTAAAGGGTG OAS1 (Rev) (SEQ ID NO: 8) CCGGCGATTTAACTGATCCTGOAS2 (Fwd) (SEQ ID NO: 9) AGGTGGCTCCTATGGACGG OAS2 (Rev) (SEQ ID NO: 10)TTTATCGAGGATGTCACGTTGG MX1 (Fwd) (SEQ ID NO: 11) CAGCACCTGATGGCCTATCAMX1 (Rev) (SEQ ID NO: 12) ACGTCTGGAGCATGAAGAACTG GAPDH (Fwd)(SEQ ID NO: 13) TTCGACAGTCAGCCGCATCTTCTT GAPDH (Rev) (SEQ ID NO: 14)CAGGCGCCCAATACGACCAAATC

The ΔΔCq method was used to calculate changes in mRNA expression levelsrelative to the indicated controls.

C-Circle Assay

The protocol for C-circle amplification was slightly modified from thatpublished by Henson et al. (Nat Biotechnol 27(12):1181-1185, 2009).Briefly, genomic DNA was purified, digested with AluI and MboI andcleaned up by phenol-chloroform extraction and precipitation. DNAconcentration was measured using a NANODROP™ and diluted in ultracleanwater. Final template amount of 100, 50 or 25 ng was diluted to 10 μlwith water and combined with 10 μl of 0.2 mg/ml BSA, 0.1% Tweendetergent, 1 mM each dATP, dGTP, dTTP and 1×Φ29 Buffer (NEB) in thepresence or absence of 7.5U ΦDNA polymerase (NEB). Samples wereincubated at 30° C. for 8 hours and then at 65° C. for 20 minutes. Thereaction products were diluted to 100 μl with 2× SSC and dot-blottedonto a 2× SSC-soaked nylon membrane. DNA was UV cross-linked onto themembrane and hybridized with a ³²P end-labelled (CCCTAA)₄ oligo probe(SEQ ID NO: 15). Blots were washed in 2× SSC and 1× SSC/0.1% SDS andexposed to PhosphorImager screens, scanned and quantified using aTyphoon 9400 PhosphorImager (Amersham/GE Healthcare).

Immunofluorescence and IF-Telomere FISH

For all immunofluorescence experiments, cells were grown on glass coverslips in 24-well plates. At the time of harvest the cells were washedtwice with PBS and fixed with 2% paraformaldehyde in PBS for 10 minutesat room temperature. Following fixation, the cells were washed twicewith MilliQ water and permeabilized with KCM buffer (120 mM KCL, 20 mMNaCl, 10 mM Tris pH 7.5 and 0.1% TritonX-100) for 10 minutes at roomtemperature. Cells were washed twice with PBS and stored at 4° C.

Fixed and permeabilized cells were incubated for 30 minutes in blockingbuffer (PBS with 5% normal goat serum, Jackson ImmunoResearch cat #005-000-121), 0.3% fish skin gelatin (Sigma cat # G7765). Blockingbuffer was removed and primary antibodies diluted in blocking bufferwere added and incubated for 1 hour at room temperature. Primaryantibodies were removed and the cells were rinsed quickly with PBSfollowed by 4,5-minute washes in PBS. After the final wash, the coverslips were incubated with secondary antibody diluted in blocking bufferfor 30 minutes. The cover slips were washed as before andcounter-stained with Hoescht DNA dye. Cells were rinsed with MilliQwater to remove excess salt and mounted with ProLong Gold antifade (LifeTechnologies cat # P36930). Mounted cover slips were left in the darkovernight at room temperature to allow for the mounting medium to cure,following which they were imaged on a Zeiss LSM 780 confocal microscopeat 63× magnification.

For immunofluorescence combined with telomere FISH, cover slips weretreated as described above with the following modifications. The initial30 minute incubation in blocking buffer was carried out at 37° C. in thepresence of 100 μg/mL RNaseA. Following the incubation with secondaryantibody and washing in PBS, the coverslips were fixed in 2%paraformaldehyde for 10 minutes at room temperature then washed threetimes with MilliQ water. The cells were ethanol dehydrated by sequentialincubation in 70% ethanol for 3 minutes, 90% ethanol for 2 minutes and100% ethanol for 2 minutes and allowed to air dry for 15 minutes. FiveμL of a 0.3 μg/mL stock of the Alexa488-PNA-TelC (PNA Bio cat #F1004)was spotted onto a clean glass slide and coverslips were placed cellsside down onto PNA probe solution. The slide was heated was heated to74° C. for 5 minutes then placed in a humidified chamber and allowed tohybridize overnight. Coverslips were washed 3 times for 5 minutes in PNAwash buffer A (70% formamide and 10 mM Tris pH 7.5), 3 times for 5minutes in PNA wash buffer B (50 mM Tris pH7.5, 150 mM NaCl and 0.8%Tween-20) and then rinsed twice with MilliQ water. Hoescht was added tothe second wash with PNA wash buffer B. Coverslips were air-dried andmounted using ProLong Gold.

Antibodies

PML (cat # ABD-030) was from Jena Bioscience and used at a 1:1000dilution. ICP4 was tissue culture supernatant generated from the 58-Shybridoma clone from American Type Culture Collection (cat # HB-8183)and used at a 1:100 dilution. ASF1A (cat # 2990) and ASF1B (cat # 2769)were both from Cell Signaling Technology and used at 1:1000 dilution.HP1 antibody was from Upstate and used at a 1:100 dilution. Goatanti-mouse Alexa Fluor 488 (cat # A11029), goat anti-rabbit Alexa Fluor555 (cat # A21429) and goat anti-mouse Alexa Fluor 647 (cat # A21236)were all from Life Technologies and used at a 1:1000 dilution.

Example 2 ALT-Dependent Replication of ICP0 Null HSV-1

This example describes the finding that ICP0-deficient HSV-1 selectivelyreplicates in and kills ALT-dependent tumor cells.

Localization of HSV-1 Viral Genomes Resembles ALT Generated ECTRs

There is a striking overlap between the molecular targets of tumormutations and DNA virus proteins (O'Shea, Oncogene 24, 7640-7655, 2005).Herpes simplex virus 1 (HSV-1) is an enveloped ˜152 kbp double-strandlinear DNA virus that undergoes lytic replication in a wide range ofcell types (Knipe, D. M. & Howley, P. M. Fields virology. 6th edn,(Wolters Kluwer/Lippincott Williams & Wilkins Health, 2013). Earlypost-entry, the circularized viral genomes accumulate within PML nuclearbodies (PML NBs) along the periphery of the nuclear envelope. Thisaccumulation is demonstrated by immunofluorescence for PML protein andthe HSV-1 encoded ICP4 protein, which binds and marks viral genomes(FIGS. 1A and 1B). If left unchecked, this association results in thesequestration of viral genomes by PML NBs resulting in inhibition ofviral replication and triggering of the expression of cellular genesthat induce a protective antiviral and host immune response (FIG. 1A)(Everett et al., J Virol 80, 7995-8005, 2006; Boutell, J Gen Virol 94,465-481, 2013). To prevent this, HSV-1 encodes a viral protein, ICP0,which degrades PML NBs as well as blocks the activation of downstreamantiviral genes (FIG. 1B) (Boutell and Everett, J Gen Virol 94, 465-481,2013).

Mutant viruses that lack ICP0 (HSV-1 ΔICP0) are highly defective andexhibit ˜1000-fold defect in replication efficiency in normal cells atlow multiplicities of infection (MOI) as measured by plaque assay (FIG.1E). This defect in replication can be overcome at high MOI, suggestingthe restriction is saturable (FIG. 1E). Furthermore, it has beenobserved that an ICP0 deleted virus replicates to wild-type levels inthe U2OS osteosarcoma derived cell line at low MOI (FIG. 1F) (Yao andSchaffer, J Virol 69, 6249-6258 , 1995). This indicates that potentialrestriction factor(s) may have been lost in these cells duringtransformation. The loss of critical HSV-1 specific restriction factorsis further supported by somatic cell hybridization experiments in whichlytic replication of HSV-1 ΔICP0 is inhibited after U2OS are fused withthe non-permissive HEL fibroblasts (Hancock and Corcoran, Virology 352,237-252, 2006). However, it is unknown what those factor(s) are and whyU2OS cells rescue replication of HSV-1 ΔICP0 back to wild type levels.

It was hypothesized that the rescue of HSV-1 ΔICP0 replication by U2OScells is due to the use of ALT as the primary telomere maintenancemechanism in these cells. Although the molecular understanding of ALT iscurrently limited, there are two defining characteristics of ALT thatbare striking resemblance to HSV-1 infection. First, telomeremaintenance via ALT generates a large amount of extrachromosomaltelomeric repeat (ECTR) DNA. This DNA is highly repetitive and presentin the nucleus as double-stranded linear, double-stranded circular aswell as partially double-stranded circular forms (FIG. 1C) (Nabetani andIshikawa, Mol Cell Biol 29, 703-713, 2009; Henson et al., Nat Biotech27, 1181-1185, 2009). Second, together with telomeres, ECTRs accumulatein and co-localize with PML NBs to form ALT-associated PML NBs (APBs)(Yeager et al., Cancer Res 59, 4175-4179 , 1999). APBs are typicallypresent in 5% of asynchronously dividing ALT (Yeager et al., Cancer Res59, 4175-4179 , 1999) cells and can be visualized via immunofluorescenceof cells using ALT, such as U2OS, stained for PML and a PNA-TelC FISHprobe (FIG. 1D). Traditionally they have been characterized by theirlarge size (>1 μM) and the presence of telomere interacting proteins andDNA damage response proteins in addition to telomeres and ECTRs (Chunget al., Nucleus 3, 263-275, 2012). However, a few groups have reportedthe presence of smaller APBs in 50-90% of asynchronously dividing ALTcells (Nabetani et al., J Biol Chem 279, 25849-25857, 2004; Osterwald,Biotechnol J 7, 103-116, 2012).

The current model of ALT suggests that PML NB s facilitate telomeremaintenance as well as sequesters ECTRs. It has been proposed that thesequestration of ECTRs by PML NBs prevents the ends of the linear doublestrand DNA ECTRs from being recognized as double strand breaksactivating a deleterious DNA damage response (FIG. 1C) (Chung et al.,Nucleus 3, 263-275, 2012). However, this model assumes accumulation ofECTRs in PML NBs is inert and fails to address the potential long-termimpact that such an accumulation of DNA would have on the normalfunctions of PML NBs. Notwithstanding, the association of ECTRs with PMLNBs presents an intriguing convergence with viral infection anddetection of their genomes.

Under normal conditions, organization of these bodies is dependent onPML. In addition to PML a myriad of other proteins have been reported tolocalize to these nuclear domains, such as ATRX, DAXX and SP100.Typically, 1-30 PML NBs, depending on cell type and phase of the cellcycle, are observed at the inter-chromatin space (Bernardi and Pandolfi,Nat Rev Mol Cell Biol 8, 1006-1016). There, PML NBs act as hubsregulating chromatin organization, transcription (activation andrepression), sequestration of foreign DNA, DNA repair, cytokine andinterferon signaling, and senescence among many other diverse cellularprocesses (Bernardi and Pandolfi, Nat Rev Mol Cell Biol 8, 1006-1016;2007; Sahin et al., J Pathol 234, 289-291, 2014; Maarifi et al.,Cytokine Growth Factor Rev 25, 551-561, 2014; Batty et al., Front Biosci14, 1182-1196 , 2009). Since PML NBs are at the nexus of telomeremaintenance via ALT and HSV-1 infection and present in limited number,it was hypothesized that the association of telomeres and ECTRs with PMLNBs could saturate their capacity to sequester extrachromosomal DNA.This would in essence render PML NBs blind to incoming HSV-1 viralgenomes at low MOI. If true, one would expect that in ALT cells, such asU2OS, incoming viral genomes would be free from the constraints andsequestration imposed on them by PML NBs. From this two testablepredictions arise: disruption of PML NBs in a non-ALT cell should rescuereplication of HSV-1 ΔICP0, and incoming viral genomes would fail tolocalize with PML NBs in an ALT cell.

Using RNAi, PML NBs were disrupted by knocking down PML, ATRX or DAXX inBJ fibroblasts (non-ALT) (FIG. 5A). Lytic replication of HSV-1 WT orΔICP0 was then measured by plaque assay in the presence of the knockdownand compared to lytic replication efficiency in BJ cells that receivedcontrol siRNA. In agreement with previous reports (Everett et al., JVirol 80, 7995-8005, 2006; Lukashchuk, J Virol 84, 4026-4040, 2010;Boutell, J Gen Virol 94, 465-481, 2013), disruption of the PML NB viaknockdown of its individual components resulted in only a modest, 5- to10-fold rescue in replication of an ICP0 deleted HSV-1 virus (FIG. 5B).Although others have shown that simultaneous knockdown of variouscomponents of PML NB such as PML, Sp100 and DAXX yields nearly a100-fold rescue in lytic replication of HSV-1 ΔICP0 in non-permissivecells (Glass and Everett, J Virol 87, 2174-2185, 2013), this is stillfar short of the 1000-fold defect in lytic replication seen in primarycells when compared to WT HSV-1 (FIG. 1E). This indicates that althoughPML NBs play a role in suppression of HSV-1 ΔICP0 lytic replication,there are other cellular factors that contribute to the suppression ofHSV-1 ΔICP0. Furthermore, U2OS cells have likely acquired multiplemutations that result in the rescue of lytic replication of an ICP0deleted HSV-1 virus back to wild type levels.

While these data demonstrate that PML NB s are in part responsible forthe restriction of HSV-1 replication, they do not delineate betweenrestriction due to sequestration and/or restriction due to modificationof the viral genome by PML NBs. It was reasoned that if sequestration ofHSV-1 viral genomes is sufficient to suppress lytic replication of anICP0 deleted virus in U2OS cells, then one would expect a lack ofco-localization between PML NBs and HSV-1 viral genomes. Whenimmunofluorescence was performed for PML, ICP4 and PNA-TelC in U2OScells, along the edge of plaques it was observed that viral genomesstill associate with PML NBs (FIG. 1G). In addition, association betweentelomeric repeat DNA and PML NBs in infected U2OS cells was observed,and in some cases, viral genomes, telomeric repeat DNA and PML NBs allco-localized. Taken together, these data indicate that the presence ofECTRs and APBs does not prevent the association of PML NBs with incomingviral genomes. This indicates that the association of HSV-1 viralgenomes with PML NBs is not sufficient to restrict HSV-1 replication butthat additional modifications to the viral genome or activation ofdownstream signaling events is required for restriction of lyticreplication at low MOI by PML NBs.

Cells that use ALT for TMM Fail to Transcriptionally Respond to HSV-1Infection

Because HSV-1 genomes co-localize with PML NBs in U2OS cells with noadverse effect on viral lytic replication, yet co-localization innon-ALT cells results in restriction, it was reasoned that a fundamentalchange in the PML NB has occurred in U2OS cells. Furthermore, sincedisruption of PML NBs via RNAi fails to restore replication of an ICP0deleted virus back to wild type levels, it suggests that U2OS cells mayharbor additional alterations that alleviate restriction of HSV-1 ΔICP0lytic replication. In addition to the disruption of PML NBs, ICP0 is apotent transactivator of both viral and cellular gene expression duringHSV-1 infection (Everett, EMBO J 3, 3135-3141, 1984). It washypothesized that changes to the cellular transcriptional program uponinfection may cooperate with PML NBs to restrict HSV-1 ΔICP0 lyticreplication. Loss or alterations to both the PML NB s and cellulartranscriptional program in U2OS cells may explain the rescue of lyticreplication of an ICP0 deleted HSV-1 virus observed in these cells.

In order to define the ICP0 induced cellular transcriptional programduring HSV-1 infection, a series of RNA-seq and microarray experimentswere performed. Since tumor derived cell lines carry large numbers ofmutations that may convolute downstream gene expression analysis andpotentially affect HSV-1 infection, normal primary small airwayepithelial cells (SAEC) were used to obtain a baseline gene expressionprofile to which all subsequent gene expression profiles would becompared. SAEC were infected with HSV-1 WT or HSV-1 ΔICP0 and total RNAwas collected at 8 hours post-infection (hpi). Significantlydifferentially expressed genes between SAEC infected with HSV-1 ΔICP0 vsSAEC infected with HSV-1 WT were determined using a threshold q-value≥0.05 and a Log2 fold change ≥0.8 or ≤−0.8 (FIG. 2A). By these criteria,1047 cellular genes were transcriptionally activated (blue) and 1259cellular genes were transcriptionally suppressed (red) by ICP0 in thecontext of HSV-1 infection (FIG. 2A). Those genes suppressed by ICP0(red) were subjected to a hypergeomteric test using the MolecularSignatures Database (MSigDB) in order to calculate overlap with thehallmark gene set. Those genes suppressed by ICP0 showed a significantoverlap with genes that are upregulated in response to pro-inflammatoryand interferon stimuli. Additionally, downstream targets of p53, cellcycle regulators such as E2F targets and components of the G2/Mcheckpoint, as well as genes down-modulated in response to UV, were allsuppressed by ICP0 during HSV-1 infection (FIG. 2B). A similar analysisin MSigDB using the curated pathways gene sets, which includes canonicalsignaling pathways from Biocarta, Kegg, Reactome among others, revealeda significant overlap between ICP0 suppressed genes and genes involvedin the regulation of telomerase activity (FIG. 2C). Taken together thesedata begin to assemble a gene signature established during infectionthat has elements that overlap with pro-inflammatory, interferon, cellcycle, DNA damage signatures as well as telomere maintenance, all ofwhich cooperate to prevent HSV-1 lytic replication in the absence ofICP0.

It was reasoned that comparing the cellular transcriptional programestablished by ICP0 in normal primary SAEC to the cellulartranscriptional program established by ICP0 in permissive U2OS cellswould allow identification of those genes or pathways that cooperatewith PML NBs to restrict an ICP0 deleted HSV-1 virus. Furthermore, itwas believed these studies could provide further insights into how thissignature is altered not only in U2OS cells but also in other cell typesthat use ALT to maintain telomere length. To this end, U2OS cells wereinfected with HSV-1 WT or HSV-1 ΔICP0 virus and RNA was collected at 12hpi, which was subjected to gene expression analysis on an affymetrixGeneChip human gene ST 1.0 microarray. Significantly differentiallyregulated genes were determined using an adjusted p-value ≥0.05 and Log2fold change ≥0.8 or ≤−0.8 (FIG. 2D). In striking contrast to 2306differentially regulated genes in SAEC infected cells, 139 genes weretranscriptionally activated and only 31 genes were transcriptionallysuppressed by ICP0 in U2OS cells in the context of HSV-1 infection.Moreover, when ICP0 suppressed genes from SAEC and U2OS were compared,they exhibited very little overlap, indicating that those genes that aredifferentially regulated in U2OS are not likely to contribute to therestriction of HSV-1 ΔICP0 (FIG. 2E). Given the small number of genesdifferentially regulated between HSV-1 ΔICP0 and HSV-1 WT in U2OS cells,coupled with the observed rescue of HSV-1 ΔICP0 lytic replication inthese cells suggests that the transcriptional program established byICP0 during HSV-1 infection in SAEC is genetically phenocopied in U2OScells. In addition, somatic cell hybridization experiments havedemonstrated that the rescue of HSV-1 ΔICP0 lytic replication in U2OS islikely to due to the loss of one or more cellular restriction factors(Hancock et al., Virology 352, 237-252, 2006). This argues that genes orpathways suppressed by ICP0 during HSV-1 infection are the criticalfactors that act to restrict viral replication and not the ICP0transcriptionally upregulated genes.

PML NBs exhibit anti-viral activity towards HSV-1 as well as severalother viruses (Everett, Oncogene 20, 7266-7273, 2001). Given theatypical presence of ECTR DNA in ALT cells that associates with and issequestered by PML NBs, similar to HSV-1 viral genomes, it washypothesized that the ECTRs may also trigger other anti-viral pathways.Since these pathways would be restrictive to both viral replication andcell growth they would have to be turned off or down-modulated duringtransformation and the transition to ALT. If true, then this wouldexplain the lack of activation of the restrictive transcriptionalprofile in U2OS cells that is observed in HSV-1 ΔICP0 infected SAEC(FIG. 2). In addition, if an anti-viral state is initiated in cellstransitioning to ALT, which must then be lost, then other cells ortumors that use ALT should also fail to activate the restrictivetranscriptional profile upon infection with HSV-1 ΔICP0.

Total RNA from either the osteosarcoma derived cell line SAOS2 or thebreast cancer derived cell line SKBR3 infected with either HSV-1 WT orHSV-1 ΔICP0 was prepared and analyzed by microarray as described above.Applying the same thresholds used for SAEC and U2OS, only 1 gene wassignificantly differentially expressed between SAOS2 HSV-1 ΔICP0infected cells and SAOS2 HSV-1 WT infected cells (FIG. 2F). A similaranalysis of SKBR3 HSV-1 ΔICP0 infected cells compared to SKBR3 HSV-1 WTinfected cells showed only 47 ICP0 transcriptionally activated genes(blue) and 10 ICP0 transcriptionally suppressed genes (red) (FIG. 2G).This further supports the hypothesis that the transition to ALT leads tothe inability of these cells to mount an appropriate restrictiveanti-viral response.

In contrast, tumor derived cell lines that use telomerase as theirpreferred telomere maintenance mechanism (TMM) should behave moresimilar to the normal primary SAEC cells and activate a similarrestrictive anti-viral transcriptional response when infected with HSV-1ΔICP0 compared to HSV-1 WT. The telomerase-positive osteosarcoma derivedcell lines HOS and SJSA1 were infected with either HSV-1 ΔICP0 or HSV-1WT virus as above and RNA was harvested for gene expression analysis bymicroarray. Significantly differentially regulated genes were determinedusing an adjusted p-value ≥0.05 and Log2 fold change ≥0.58 or ≤−0.58 forHOS cells and Log2 fold change ≥1 or ≤−1 for SJSA1. The resultsdemonstrated that 1564 genes were differentially regulated in HOS and4265 in SJSA1 cells with 1120 genes in HOS and 3359 genes in SJSA1 beingtranscriptionally suppressed by ICP0. Furthermore, when the overlapbetween the ICP0 transcriptionally suppressed genes was computed usingthe hallmark gene sets from MSigDB (as described above), there was anoverlap with many of the same gene sets observed when the same analysiswas performed on the SAEC expression data. There is a loss in someaspects of the inflammatory and interferon signaling gene sets from boththe HOS and SJSA1 expression data sets when compared to SAEC. However,this is not too surprising since tumor cells in general have beenreported to be less responsive to interferon signaling and establishmentof an anti-viral state (Hummel et al., Mol Ther 12, 1101-1110, 2005).Furthermore, since tumor cells tend to be more permissive to replicationof HSV-1 ΔICP0, regardless of TMM, when compared to normal primary cellsit is possible that a partial loss of the anti-viral transcriptionalprogram as seen here may account for this partial rescue. Takentogether, these data demonstrate that cells that use ALT fail to respondand initiate an anti-viral transcriptional response as seen in normalprimary SAEC as well as telomerase positive tumor cell lines.

ALT Cells Fail to Initiate Anti-Viral Response when Challenged byIntracellular DNA

The data described herein predicts that ALT cells should have adiminished capacity to transcriptionally upregulate interferonstimulated genes (ISG) in response to pathogenic stimuli. Activation ofISGs can occur through extrinsic initiated or intrinsic initiatedpathways (Ivashkiv and Donlin, Nat Rev Immunol 14, 36-49, 2014;Schneider, Annu Rev Immunol 32, 513-545, 2014). To test whether thepreferred TMM of a cell influences its ability to initiate anappropriate transcriptional response via the extrinsic pathway, a panelof ALT and non-ALT cell lines were treated with varying amounts ofinterferon-α. Fours hours post addition of IFNα, RNA was collected andthe transcriptional induction of several downstream ISGs (IFIT1, IFIT2,IFIT3, OAS1, OAS2 and MX1) were monitored by RT-qPCR as a read out forsensitivity to IFNa. Both telomerase-positive and ALT cells were foundto be equally capable of transcriptionally inducing ISGs in response totreatment with IFNα (FIGS. 3A and 6A).

However, frequently a cell must recognize and respond to invadingpathogens from within. This occurs through the detection a pathogensproteins and/or nucleic acids (Schneider et al., Annu Rev Immunol 32,513-545, 2014; Ivashkiv and Donlin, Nat Rev Immunol 14, 36-49, 2014).Once detected, the cell must mount an appropriate response to containand limit spread of the pathogen. This typically occurs through theactivation of ISGs capable of limiting viral replication and alertingsurrounding cells to the potential threat. It was hypothesized that theECTRs generated during ALT may be mistaken as pathogenic DNA activatinga cellular anti-viral response. Prolonged activation of such a responsewould be deleterious to the cell and would have to be dampened for thecell to fully transition to ALT. Although the response to IFNs in cellsdependent on ALT was found to be still intact, it was reasoned that itsability to respond to an internally detected threat might becompromised. To test this, an empty pCDNA3.1 plasmid was introduced intothe cell via transfection in order mimic DNA from an invading pathogen,such as HSV-1. RNA was harvested from the cells six hourspost-transfection and subjected to RT-qPCR analysis for downstream ISGsas described above. Those cells that use ALT to maintain telomere lengthexhibited a severely dampened response to plasmid DNA (FIGS. 3B and 6B).However, cells that use telomerase largely induce the expression ofdownstream ISGs as would be expected (FIGS. 3B and 6B). It wasquestioned whether this response was specific to circular plasmid DNA orif other forms of extrachromosomal DNA could initiate a similarresponse. To test this, purified and sheared calf thymus DNA wastransfected and activation of ISG effectors was assayed (FIG. 3B). Theresults demonstrated that the lack of response to foreignextrachromosomal DNA in ALT cells was a general phenomenon regardless ofform or source.

Next, studies were performed to determine whether infection with HSV-1would recapitulate the differential response of telomerase-positive andALT-dependent cells when challenged with foreign DNA. Both HOS (tel+)and U2OS (ALT) were infected with either HSV-1 WT or HSV-1 ΔICP0 at anMOI of 1 and incubated for 4 hours. Following treatment with the virus,RNA was harvested from the infected and uninfected cells and subjectedto RT-qPCR as described above. HOS cells infected with HSV-1 ΔICP0exhibited activation of many of the ISG assayed, while U2OS cellsinfected with HSV-1 ΔICP0 failed to induce transcription of the sameISGs (FIG. 3C). As expected, infection with HSV-1 WT led to thesuppression of ISGs transcription in both HOS and U2OS. It was alsoobserved that the induction of the ISGs assayed in response to HSV-1ΔICP0 infection was not as robust as with IFNa treatment or transfectionof DNA. This is likely due to the redundant anti-viral activity of otherimmediate early HSV-1 genes that are still expressed by HSV-1 ΔICP0,albeit to a lesser extent. Importantly, since HSV-1 deposits the viralgenome directly into the nucleus this suggests that the response that isinitiated is a result of the detection of the viral DNA in the nucleusand not in the cytoplasm where most DNA sensors have been proposed tofunction.

Since many of these ISGs can also be upregulated in response todouble-stranded RNA via the toll-like receptor 3 (TLR3), it was examinedwhether this defect in ALT cells is specific to DNA or extends toforeign RNA as well. To test this, HOS (Tel+) and U2OS (ALT) cells weretransfected with the dsRNA mimic PolyI:C. Four hours post-transfection,RNA was harvested from these cells and subjected to RT-qPCR as before.Although ALT cells failed to respond to extrachromosomal DNA, theirability to mount an appropriate transcriptional response to dsRNA wasstill intact (FIG. 3D).

Taken together, these data indicate that cells that use ALT as theirpreferred TMM have lost the ability to initiate and propagate anappropriate anti-viral transcriptional response when challenged withextrachromosomal DNA from within. The RNA-seq and microarray analysisfurther demonstrated that while this response overlaps with some of thesame targets as the interferon response, it also has elements thatoverlap with inflammatory cytokine signaling, p53, cell cycleregulation, DNA damage response and telomerase signaling. Collectively,these elements come together to generate a potent extrachromosomal DNAinduced anti-viral state in the cell. The inventors have termed thisanti-viral response the ‘Viral and Extrachromosomal DNA transcriptionalresponse’ or VECTR.

ALT, gC and ICP0 Functionally Converge

Loss of the protective anti-viral VECTR response is not only criticalfor the transition to ALT but indicates that ALT cells may be moresusceptible to incoming pathogens than non-ALT cells. Since ICP0 and ALTseem to overlap in their functions, this may explain why U2OS cellsrescue replication of an ICP0 null virus. Given these results, theprediction is that cells that use ALT to maintain telomere length shouldalso rescue replication of an ICP0 deleted HSV-1 virus. This was testedby performing a plaque assay on 16 non-ALT and 11 ALT cell lines withboth HSV-1 WT and an ICP0 deleted virus. Relative plaque formingefficiency of HSV-1 ΔICP0 virus was calculated by comparing the numberof plaques formed at a particular MOI (˜30 plaques/well) to the numberof plaques formed in WT virus at that same MOI. No non-ALT cell linesrescued replication of HSV-1 ΔICP0, yet nearly all of the ALT cell linesrescued replication to some degree (FIG. 4A, FIG. 9A, and FIG. 9B).Those cell lines that rescue to a lesser extent are all cell lines thatwere in vitro transformed. This suggests that there may be additional invivo selection that occurs, which further renders ALT cells permissiveto HSV-1 replication.

Furthermore, a widely used ICP0 loss mutant HSV-1 (strain name d11403)was found to have an unexpected mutation in its glycoprotein C gene,which is a deletion of 186th cytosine in its coding sequence (see FIG.10). As a result, glycoprotein C peptide in dICP0 virus has extensivesequence change as well as premature termination at 175th codon.

SEQ ID NO: 17 shows the nucleotide sequence of WT HSV-1 gC fromnucleotides 181-200:

(SEQ ID NO: 17) ACCCCCACATCGACCCCAAA.SEQ ID NO: 17 shows the mutant gC sequence from HSV-1 strain d11403,from nucleotides 181-199:

(SEQ ID NO: 18) ACCCCACATCGACCCCAAA.SEQ ID NO: 19 shows the amino acid sequence of WT HSV-1 gC from aminoacid 60-70:

(SEQ ID NO: 19) VTPTSTPNPNN.SEQ ID NO: 20 shows the amino acid sequence of HSV-1 strain d11403 gCfrom amino acid 60-70:

(SEQ ID NO: 20) VTPHRPQTPTM.SEQ ID NO: 21 shows the amino acid sequence of WT HSV-1 gC from aminoacid 171-180:

(SEQ ID NO: 21) PAPDLEEVLT.SEQ ID NO: 22 shows the amino acid of HSV-1 strain d11403 gC from aminoacid 171-174:

(SEQ ID NO: 22) RLPT.

This loss-of-function mutation of gC (glycoprotein C) in dICP0 HSV-1 isbelieved to inhibit its invasion efficiency into the cell because gC isa major cell membrane receptor binding protein, compared to itshomologous proteins, gA and gB. Since their binding target moleculeheparin sulfate is highly expressed in the fibroblast lineage, dICP0HSV-1 infection ability can be underestimated when infected into suchcells. At least in four osteosarcoma cell lines (HOS[TEL+], SJSA1[TEL+],SAOS2[ALT], U2OS[ALT]), the two TEL+ cell lines appear to have morefibroblast-like features, hence, presumably more heparan sulfate ontheir cell membrane, while ALT cell lines appear to be more epithelial.Thus, recombinant HSV-1 with modified gC, or gC deficient, may havehigher selectivity for ALT-dependent tumor cells relative to thesurrounding normal fibroblast tissue.

Discussion

Previous studies have shown that the loss of p53 and inactivatingmutations in ATRX, a component of the PML NB, strongly correlates withALT (Lovejoy et al., PLoS Genet 8, e1002772, 2012). However,inactivation of either has yet to recapitulate the phenotypes associatedwith ALT, indicating that additional changes are required for the fulltransition to ALT. The studies described herein demonstrated that theECTR DNA that associates with PML NBs plays a critical role in thetransition to ALT, thus defining another set of cellular changesrequired for telomere maintenance via ALT. From these data a modelbegins to take shape in which the transition to ALT is a multi-step andmulti-genic process where any one mutation may not be sufficient.

It has been proposed that PML NBs facilitate telomere maintenance in ALTas well as sequester ECTRs away from the rest of the cell. Thissequestration of ECTRs by PML NBs is thought to prevent the activationof a deleterious DNA damage response. However, it is proposed hereinthat the association of ECTRs and PML NBs is part of the normal cellularintrinsic immune response. As such, recognition of ECTRs as a potentialpathogen leads to the activation and establishment of an anti-viralstate in the cell. This may occur through both PML-dependent andPML-independent mechanisms. Continued activation of this response wouldprevent cellular replication. Therefore, suppression of this anti-viralresponse would have to occur if the cell were to switch to ALT andcomplete its malignant transformation (FIG. 4B).

The studies described herein determined that inhibition of thisDNA-specific anti-viral response, termed the VECTR response, phenocopiesthe activity of the HSV-1 protein ICP0 (FIG. 4B). As a result, ALT cellsfail to recognize or respond appropriately to incoming HSV-1 genomes.The result is rescue of lytic replication of an ICP0 deleted HSV-1 virusin ALT dependent tumors. However, it is interesting that the tumorderived ALT cell line rescue of HSV-1 ΔICP0 lytic replication is morepronounced than the in vitro transformed ALT cell lines. This suggestsin vivo selective pressures, which are missing when in vitro transformedcells transition to ALT, may further facilitate the transition to ALT.

Example 3 ALT-Dependent Tumor Xenograft Models

To assess oncolytic activity of HSV-1 ΔICP0 in vivo, the ALT-dependentosteosarcoma tumor cells SAOS2, which had been stably transduced with alentiviral vector expressing a CMV driven luciferase reporter gene, wereinjected into the flanks of nude mice. Growth of the xenograft tumorswas monitored by both luciferase activity and by tumor volume. Mice withestablished tumors were injected with either wild-type HSV-1 or HSV-1ΔICP0 at Day 0 (5×10⁵ PFU), Day 3 (5×10⁵ PFU) and Day 8 (1×10⁶ PFU).Tumor cell viability, as measured by luciferase activity (FIG. 7A), andtumor cell volume (FIG. 7B) were measured at Days 0, 3, 8, 14, 20, 24,27, 31 and 36. The results demonstrated that administration of either WTHSV-1 or HSV-1 ΔICP0 reduced tumor volume and eliminated tumor cellviability as demonstrated by the reduction of luciferase activity in thetumors down to background levels. In addition, it was found that HSV-1ΔICP0 had no adverse effect on the overall health of the mouse, whereasadministration of HSV-1 WT led to the death of the mouse after 8 days.

A second experiment was conducted in SAOS2 xenograft mice. Mice withestablished tumors were administered either DMEM (as a control), WTHSV-1 or HSV-1 ΔICP0 at Day 0 (5×10⁵ PFU), Day 4 (5×10⁵ PFU) and Day 8(1×10⁶ PFU). Tumor cell viability (FIG. 7C) and tumor volume (FIG. 7D)were measured at Days 0, 4, 8, 12 and 17. The results demonstrated thatadministration of either WT HSV-1 or HSV-1 ΔICP0 reduced tumor cellviability and tumor volume.

Next, A549 cells, which are Tel+ adenocarcinoma tumor cells, were usedto establish tumor xenografts in nude mice. Since A549 cells are notALT-dependent, HSV-1 ΔICP0 is replication defective in these cells. Micewith established tumors were injected with either DMEM (as a control),WT HSV-1 or HSV-1 ΔICP0 at Day 0 (5×10⁵ PFU), Day 3 (5×10⁵ PFU) and Day7 (1×10⁶ PFU). Tumor cell viability was measured at Days 0, 3, 7, 12 and16. The results demonstrated that neither WT HSV-1 nor HSV-1 ΔICP0 wereable to significantly decrease tumor viability.

Example 4 Additional Recombinant ICP0-Deficient Viruses

HSV-1 ICP0 null virus can be modified to include additional genedisruptions, such as disruptions in one or more of the ICP47 gene, theICP34.5 gene and the ICP6 gene. Recombinant HSV-1 can also be furthermodified to encode heterologous proteins, such as an immunostimulatorymolecule.

In one example, HSV-1 ΔICP0 is modified to insert a nucleic acidconstruct that includes the GM-CSF coding region into both TR_(L) andIR_(L). The GM-CSF coding sequence is operably linked to a CMV promoterand a polyA sequence (FIG. 8, construct 2). The genome sequence of thisvirus is set forth herein as SEQ ID NO: 15.

In another example, HSV-1 ΔICP0 is modified as indicated above to insertGM-CSF into both TR_(L) and IR_(L). The virus is further modified bypartial deletion of the ICP47 gene (A1-78 bp) (FIG. 8, construct 3). Thepartial deletion of ICP47 removes the first 78 bp (encoding 26 aminoacids) starting from the ATG start codon. It also removes the rest ofthe ATG sites downstream that could potentially be used as a start site.The genome sequence of this virus is set forth herein as SEQ ID NO: 16.

In one example, HSV-1 ΔICP0 is modified to insert a nucleic acidconstruct that includes the GM-CSF coding region into both TR_(L) andIR_(L). The GM-CSF coding sequence is operably linked to a CMV promoterand a polyA sequence (FIG. 8, construct 2). The virus is furthermodified by a point mutation of the gC gene (Δ168c) (FIG. 10) whichresults in non-expressing Gc. The genome sequence of this virus is setforth herein as SEQ ID NO: 23.

In another example, HSV-1 ΔICP0 is modified as indicated above to insertGM-CSF into both TR_(L) and IR_(L). The virus is further modified by apoint mutation of the gC gene (Δ168c) (FIG. 10) which results innon-expressing Gc. The virus is further modified by partial deletion ofthe ICP47 gene (Δ1-78 bp) (FIG. 8, construct 3). The partial deletion ofICP47 removes the first 78 bp (encoding 26 amino acids) starting fromthe ATG start codon. It also removes the rest of the ATG sitesdownstream that could potentially be used as a start site. The genomesequence of this virus is set forth herein as SEQ ID NO: 24.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the disclosure and should not be takenas limiting the scope of the disclosure. Rather, the scope of thedisclosure is defined by the following claims. We therefore claim allthat comes within the scope and spirit of these claims.

1. A method of treating an alternative lengthening of telomeres(ALT)-dependent cancer in a subject, comprising: selecting a subjecthaving an ALT-dependent cancer; and administering to the subject arecombinant herpes simplex virus (HSV)-1 that is infected cell protein 0(ICP0)-deficient, glycoprotein C (gC)-deficient, or both ICP0-deficientand gC-deficient, thereby treating the ALT-dependent cancer in thesubject. 2-20. (canceled)
 21. A recombinant herpes simplex virus (HSV)-1comprising a genome sequence of SEQ ID NO: 15, 16, 23 or
 24. 22. A PCRamplification reaction comprising a primer pair selected from: SEQ IDNOs: 1 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 5 and 6; SEQ ID NOs: 7and 8; SEQ ID NOs: 9 and 10; SEQ ID NOs: 11 and 12; and SEQ ID NOs: 13and 14.